Epidermal growth factor receptor (EGFR), an oncogenic receptor tyrosine kinase, is a major therapeutic target in several types of cancers. Two EGFR monoclonal antibodies (mAbs) are used to treat EGFR- positive colorectal cancer (CRC), including cetuximab (CTX) and panitumumab, which have the same mechanism of action. Unfortunately, primary and acquired resistance to the mAbs is very common: response in only 10-20% patients and lasts typically for 3-12 months. Current approach to combating the resistance is to combine the mAb with agent(s) that targets compensatory or altered downstream signaling molecules (ErbB2, cMET, KRAS, BRAF and PIK3CA), but clinical efficacy of such approach is uncertain. Importantly, we found that hPEPDG278D, a recombinant enzymatically inactive mutant of human peptidase D, strongly inhibits the proliferation of all tested CTX-resistant CRC cell lines, and silences EGFR, ErbB2 and their downstream signaling molecules in these cells. Our results suggest that EGFR and ErbB2 are pivotal therapeutic targets in CTX-resistant CRC cells. We recently showed: a) hPEPDG278D specifically binds to the extracellular domain of EGFR and ErbB2, disrupts their signaling units and directs them for degradation via endocytosis, and therefore is a novel dual inhibitor of EGFR and ErbB2; b) it selectively inhibits cancer cells overexpressing EGFR and/or ErbB2 in vitro and in vivo; c) it does not show toxicities in mouse studies. The goal of our proposal is to further evaluate hPEPDG278D for overcoming CTX resistance and to substantiate the novel concept that EGFR and ErbB2 remain critical therapeutic targets in CTX-resistant CRC cells. We hypothesize that hPEPDG278D overcomes CTX resistance by suppressing both ErbB2 and CTX-insensitive EGFR, and propose three Aims to test the hypothesis. Aim 1 is to determine the therapeutic efficacy of hPEPDG278D against CTX-resistant CRC tumors in vivo, using both orthotoptic tumor models and patient- derived xenograft models. Oncogenic ErbB3 and ErbB4 (other EGFR members) as well as cMET are also implicated in CTX resistance in CRC. Aim 2 is to investigate whether hPEPDG278D indirectly suppresses ErbB3, ErbB4 and cMET by disrupting their heterodimerization with EGFR or ErbB2, using both cell lines and treated tumors from Aim 1. Besides targeting ErbB2, hPEPDG278D also targets both CTX-sensitive and -insensitive EGFR. Aim 3 is to determine the mechanisms by which hPEPDG278D targets CTX-insensitive (mutated or wild type) EGFR in relevant CRC cell lines. In summary, it is a widely accepted concept in the field that CRC resistance to CTX and other EGFR inhibitors results largely from activation of other signaling proteins (ErbB2, cMET, KRAS, BRAF or PI3K). We expect to show that the main reason for resistance to CTX is the insensitivity of EGFR to CTX along with ErbB2 overexpression; the impact of this paradigm- shifting concept on translational studies on EGFR-positive cancers should be significant. Findings from the project may also lay a solid foundation for clinical evaluation of hPEPDG278D against CRC and other cancers.